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A method for improving the efficiency of homologous recombination in cho cells and its related products and applications

A homologous recombination and cell technology, applied in the field of biomedicine, can solve the problems of low homologous recombination efficiency and no obvious improvement in homologous recombination efficiency, and achieve the effects of good condition, improved efficiency, and increased expression

Active Publication Date: 2022-06-21
ACADEMY OF MILITARY MEDICAL SCI
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  • Description
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AI Technical Summary

Problems solved by technology

[0005] Recently, relevant researchers have demonstrated through experimental studies that the simultaneous knockout of the LIG4 gene and the POLQ gene in mouse embryonic stem cells and human B lymphocytic leukemia cells (Nalm6 cells) can inhibit the NHEJ repair pathway of host cells and thereby inhibit random integration. , to improve the efficiency of homologous recombination in cells, but knocking out the POLQ gene alone in this study did not significantly improve the efficiency of homologous recombination
Many previous studies have shown that CHO cells are a cell line with very low homologous recombination efficiency. At present, the introduction of foreign gene expression in this cell is mediated by random integration and recombinase-mediated site-specific recombination technology.

Method used

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  • A method for improving the efficiency of homologous recombination in cho cells and its related products and applications
  • A method for improving the efficiency of homologous recombination in cho cells and its related products and applications
  • A method for improving the efficiency of homologous recombination in cho cells and its related products and applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] Example 1 Construction of the CRISPR / Cas9 vector of the POLQ gene and its gene editing efficiency verification

[0092] 1. Experimental materials

[0093] CHO-K1( CCL-61 TM ) cells were purchased from ATCC; DMEM F12 medium, phosphate buffer, and 0.25%-EDTA trypsin were purchased from Gibico; fetal bovine serum was purchased from Sijiqing; px330 and ploxp2neo were purchased from Addgene; POLQ gene amplification primers, The DNA sequence synthesis and gene sequencing of single-stranded guide RNA (sgRNA) oligonucleotide and Rosa26-SA-EGFP-polyA were completed by Shanghai Sangon Biotechnology Co., Ltd.; T4 DNA ligase, 2xQ5 Mix premix reagent, T7EI enzyme, nucleic acid Endonucleases BbsI, NotI, XhoI and SalI were purchased from NEB Company; cell genome DNA extraction kit was purchased from Tiangen Biochemical Company; chloroform, isopropanol, absolute ethanol, and normal saline were purchased from Sinopharm Chemical Reagent Co., Ltd.; Endotoxin plasmid extraction kit and...

Embodiment 2

[0112] Example 2 Construction of POLQ gene knockout CHO-K1 cells

[0113] 1. Construction of POLQ single-gene knockout mixed cell pool and screening of monoclonal cells

[0114] Press 2×10 one day in advance 5 The wild-type CHO-K1 cells were plated into six-well plates, and the high-efficiency sgRNA-px330 (4 μg) vector (sgRNA2) and pmax-GFP (1 μg) plasmid that had been constructed and verified above were selected to co-transfect CHO-K1 cells. The cells were digested and washed with FACS solution, passed through a cell mesh, and sorted by flow cytometer into a 96-well plate containing 2% double antibody and 20% serum medium. 96-well plate. On the 10th day after sorting, the wells with monoclonal cell colonies were marked under the microscope and transferred to a 6-well plate for expansion culture by trypsinization. When the confluence of cells in the wells reached more than 80%, the cells were digested, 2 / 3 cells were used for cryopreservation, and 1 / 3 of the cells were cul...

Embodiment 3

[0125] Example 3 Gene knock-in efficiency verification vector construction for ROSA26 site

[0126] 1. Experimental method

[0127] The homologous targeting backbone vector ploxp2neo (purchased from Addgene) was subjected to triple digestion with 3 μL of NotI, XhoI and SalI, followed by dephosphorylation (4 μg of digestion), and 1% agarose gel electrophoresis was used to identify the position of the enzyme-cut band. The 2.8kb band was recovered by gel; the E. coli containing Rosa26-SA-EGFP-polyA was activated for plasmid extraction, and the plasmid was double digested with 3 μL each of NotI and XhoI, and the 1.9kb fragment was recovered with 1% agarose gel, T4 After DNA ligase ligates the target fragment and the linearized vector, heat shock at 42°C for 45s and transform it into Trelief 5α competent cells. Take 100 μL of bacterial solution and spread it on LB agarose solid medium containing ampicillin resistance, and culture at 37°C overnight. The next day, 5 colony clones we...

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Abstract

The invention discloses a method for improving the efficiency of homologous recombination in CHO cells and related products and applications. The invention firstly discovers and proves that knocking out the POLQ gene alone in CHO cells and inhibiting the al-NHEJ repair pathway can efficiently improve the efficiency of CHO cells. Homologous recombination efficiency, the constructed POLQ gene-knockout CHO cells can improve the efficiency of screening stable cell lines when expressing foreign antibodies, and further increase the expression of products, and have a good effect in the preparation of recombinant protein biopharmaceuticals Application prospect.

Description

technical field [0001] The present invention belongs to the technical field of biomedicine, in particular, the present invention relates to a method for improving the efficiency of homologous recombination, and more particularly, the present invention relates to a method for improving the efficiency of CHO cell homologous recombination and related products and applications thereof. Background technique [0002] Chinese hamster ovary cells (CHO) are the most important cell lines for industrial production of recombinant protein biopharmaceuticals. Since Dr. Theodore T. Puck isolated the first wild-type CHO cells from adult female hamster ovary in 1957 , After several transformations, a commercial expression system based on DHFR-CHO (deficient dihydrofolate reductase) expression system and GS-CHO (deficient glutamine synthase) expression system has been gradually formed. At present, CHO cells have been the preferred host cells for the production of recombinant glycoprotein drug...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/90C07K14/47C12N5/10C12N15/113C12N15/67C12Q1/02
CPCC12N15/85C12N15/907C07K14/47C12N5/0682C12N15/113C12N15/67G01N33/5005A61K48/0008C12N2510/00C12N2800/107C12N2310/20
Inventor 王晶孙照霖王传杰王明乔春霞罗龙龙肖鹤陈国江李新颖涂凯刘金青冯健男沈倍奋
Owner ACADEMY OF MILITARY MEDICAL SCI
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